APPLICATION OF MOLECULAR TECHNICS FOR DIAGNOSIS OF VIRAL INFECTIONS

Transcription

1 APPLICATION OF MOLECULAR TECHNICS FOR DIAGNOSIS OF VIRAL INFECTIONS Hossein Keyvani Basic Diagnostic Methods in Virology Immunology and serology techniques (Antigen-Antibody Reactions) 1

2 ELISA ( Enzyme Link Immunosorbent Assay) Advantages of Elisa Reagents are relatively cheap and long shelf life Highly specific and sensitive Equipment can be inexpensive and widely available Can be use to variety of infection Safe and No radiation hazards Easy to perform and quick procedure 2

3 Elisa Disadvantages Enzyme activity may be affected by plasma constituents( Natural inhibitor) False Positives and Negatives possible, especially with mutated/altered antigen Sensitivity of Enzyme to temperature Takes 3 to 8 weeks after exposure to the viral agent WESTERN BLOT ( IMMUNOBLOT ) AND RIBA RIBA ( Recombinant ImmunoBlot Assays ) RIBA is a confirmatory method RIBA is very useful in the confirmation of Antibody results RIBA in viral diagnosis is mostly used for the confirmation of positive ELISA of HCV and HIV antibodies 3

4 RIBA Strip Interpretation Pos Pos Pos IND Neg 3+control band Caspsid NS3-1 HCV Proteins NS3-2 NS4 NS5 Recombinant ImmunoBlot Assays (RIBA) In Iran we recommend RIBA as a confirmatory test for a person who might be considered as HCV infected. The reason for this recommendation are: -Quality of ELISA kits -High cost of NAT tests 4

5 IFA Most commonly used fluorescent dyes are: Fluorescein: absorbs blue light and emits an intense yellowgreen fluorescence Rhodamine: absorbs yellow-green range and emits a deep red fluorescence Direct immunofluorescence Ag is fixed on the slide Fluorescein labeled Ab s are layered over it Slide is washed to remove unattached Ab s Examined under UV light in a fluorescent microscope The site where the Ab attaches to its specific Ag will show apple green fluorescence 5

6 Indirect immunofluorescence Indirect test is a double-layer technique The unlabelled antibody is applied directly to the tissue substrate Treated with a fluorochrome-conjugated anti-immunoglobulin serum Advantages and Disadvantages High Specificity High Sensitivity Subjective errors in reading Expensive equipment The expression of molecules can be observed directly Fixed slide can persist for long time Cross reacting Ab Improper Immune system( False Negative) 6

7 Chemiluminescence assay Enzymes for luminescent assay are AP( Alkaline phosphatase), HRP(HORSE RADISH PEROXIDASE) SUBSTRATE LUMINOL, ISOLUMINOL, LUCIFERIN Advantages Sensitive Fast emission of light Short Incubation Period Absence of toxicity Disadvantages False Positive Expensive Instrument 7

8 Molecular diagnostic PCR (Polymerase Chang Reaction) Step 1: Denature DNA At 95 C, the DNA is denatured (i.e. the two strands are separated). 8

9 Step 2: Primers Anneal At 40 C- 65 C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA Forward Primer Reverse Primer Step 3: DNA polymerase Extends the DNA chain At 72 C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3 ends of the primers Extension Extension 9

10 The next cycle will begin by denaturing the new DNA strands formed in the previous cycle Gel Electrophoresis 10

11 Nested PCR Nested PCR involves two sets of primers Nested reaction includes: 1. Outer PCR (PCR1) 2. Dilution 3. Inner PCR (PCR2) With high sensitivity and specificity Research and clinical applications AUTOMATED NESTED MULTIPLEX PCR SYSTEM BIOFIRE FILMARRAY BIOMERIEUX COMPANY 11

12 Nested PCR Disadvantages Extended time Increases the risk of contamination Real time PCR 12

13 REAL TIME PCR 5 NUCLEASE ASSAY USING TAQMAN PROBES Forward Primer 5 3 FAM, VIC R TaqMan Probe Q TAMRA dyes Reverse Primer PCR specificity (primer) Hybridization specificity (probe) 5 Nuclease Assay using TaqMan probes Cleavage of probe by 5 nuclease activity of Taq polymerase FRET disabled, generation of reporter signal R Q

14 5 Nuclease Assay using TaqMan probes 5 3 R Q Fluorescence Resonance Energy Transfer (FRET) from high energy to low energy dye No reporter signal with intact probe 3 2,5 2 1,5 1 0, ,5 14

15 3 2,5 2 1,5 1 0, ,5 3 2,5 2 1,5 1 0, ,5 15

16 3 2,5 2 1,5 1 0, ,5 3 2,5 2 1,5 1 0, ,5 16

17 3 2,5 2 1,5 1 0, ,5 3 2,5 2 1,5 1 0, ,5 17

18 COBAS TaqMan Lower Limit of Quantification: 6 IU/Ml Upper Limit of Quantification: 100,000,000IU/mL Conversion Factor: 5.82 Advantages of Real Time PCR It does not required gel preparation for visualization It is not time consuming compare to conventional PCR Amplification can be monitored in Real Time Possibility of Confirmation of specific amplification by Melting point analysis 18

19 AUTOMATED NESTED MULTIPLEX PCR SYSTEM BIOFIRE FILMARRAY BIOMERIEUX COMPANY Integrates sample preparation, amplification, detection and analysis. REVERS DOT BLOLT Able to detect different pathogens in one strip Able to differentiate different genotypes Able to detect mutants Mostly used for genotyping and mutant detection 19

20 REVERSE DOT BLOT METHOD Alkaline Phosphatase Strepavidin Biotin Biotin-labeled 5 NC region of amplified target 3' Genotype-specific DNA probes on nitrocellulose strip 20

21 Lab-on-a-Chip Microarray Oligonucleotides microarray: hybridization of the targets( labeled nucleic acids of RNA or DNA with fluorescent dyes) and the probes(several individual nucleic acid species immobilized on a solid surface in the spots form 21

22 Oligonucleotides microarray Sample preparation Extraction RNA or DNA Amplification with dntp labelled with fluorescent Cyanine dyes (i.e., Cy3 and Cy5) Denatured PCR products Hybridized target products with specials probe Spotted on a glass slide. Microarray software Green spots Red spots Yellow spots Up regulated genes. Down regulated genes Equal regulated genes Microarray-based Genotyping and drug-resistant HBV mutations 22

23 Protein microarray: Protein Protein interaction Prepared by immobilizing proteins(antigen or Antibodies) onto a glass slide Screened for protein ability to bind other protein in a complex,antibody's,enzymes. Protein microarray 23

24 Advantages Provide data from thousands of genes One experiment instead of many Fast and easy to obtained results Specific and Sensitive Need less volume sample and reagent. Microfluidics Technology process small quantities of fluids by using tiny channels having dimensions at the microscale typically tens to hundreds of micrometers.(10-9 to liters) 24

25 Microfluidics Technology Reducing the size will increase the volume-to surface ratio. 1. Surface tension. 2. Diffusion (allow faster transfer). 3. Fast thermal relaxation ( the larger heat distribution, especially for PCR, which requires a different system and different stages of cold and heat). 25

26 Advantages Low cost Physically small. Ease of use and compactness Reduction of human error Faster response time and diagnosis Low volume sample Real time process control and monitoring increase sensitivity. Expendable: automation and low energy consumption. 26

27 GeneXpert 27

28 DNA Sequencing - Determining the precise order of nucleotides in a pieces of DNA - DNA sequence is useful in studying fundamental biological processes and in applied fields such as diagnostic or forensic research - DNA sequencing methods have been around for 40 years, and since the mid-1970s Sequencing Methods B- Enzymatic method (Sanger, 1981) Uses dideoxy nucleotides to terminate DNA synthesis. DNA synthesis reactions in four separate tubes Radioactive datp is also included in all the tubes so the DNA products will be radioactive. With addition of enzyme (DNA polymerase), the primer is extended until a ddntp is encountered. The chain will end with the incorporation of the ddntp With the proper dntp: ddntp ratio (about 100:1), the chain will terminate throughout the length of the template. Yielding a series of DNA fragments whose sizes can be measured by electrophoresis. 28

29 Sequencing Methods Two basic methods for DNA sequencing :- A- Chemical cleavage method (Maxam and Gilbert, 1977) Base-specific cleavage of DNA by certain chemicals Four different chemicals, one for each base - A set of DNA fragments of different sizes DNA fragments contain up to 500 nucleotides Dye Terminator Sequencing A distinct dye or color is used for each of the four ddntp. Since the terminating nucleotides can be distinguished by color, all four reactions can be performed in a single tube. 29

30 Pyrosequencing 30

31 Ion Torrent After bead-based template enrichment, beads are carefully arrayed into a microtitre plate. Nucleotide species are added to the wells one at a time and a standard elongation reaction is performed. As each base is incorporated, a single H+ ion is generated as a by-product. The H+ release results in a 0.02 unit change in ph, detected by acomplementary metal-oxide semiconductor (CMOS) and an ion-sensitive field-effect transistor (ISFET) device. 31

32 Advantages Molecular Diagnostic in Pipeline Microarray integrated with SPR (Surface Plasma Resonance) 32

33 33